Assembling, Evolving and Optimizing Hybrid Synthetic Molecular Systems

  • Operon frameworks for complex synthetic systems need to be explored in a multiplexed and multilevel approach to develop design rules.
A: Several operon design elements can influence final protein expression levels.
B: E. coli expressing various fluorescent reporter protein combinations.
C: A selected set of fluorescent proteins can be quantified in an orthogonal fashion via flowcytometry.
    Operon frameworks for complex synthetic systems need to be explored in a multiplexed and multilevel approach to develop design rules. A: Several operon design elements can influence final protein expression levels. B: E. coli expressing various fluorescent reporter protein combinations. C: A selected set of fluorescent proteins can be quantified in an orthogonal fashion via flowcytometry.
  • Cell-free enzyme evolution: 
Step 1: Diversification and subsequent separation of Streptavidin DNA variants in water-in-oil droplets. Amplification of Streptavidin DNA onto magnetic beads.
Step 2: Compartmentalization of bead bound Streptavidin DNA  and subsequent production and linkage of the gene product (genotype-phenotype linkage).
Step 3: Assembly of the artificial metalloenzyme and encapsulation into water-in-oil-in-water droplets.
Step 4: Screening for highly active enzymes able to perform new-to-nature ring closing metathesis reaction.
    Cell-free enzyme evolution: Step 1: Diversification and subsequent separation of Streptavidin DNA variants in water-in-oil droplets. Amplification of Streptavidin DNA onto magnetic beads. Step 2: Compartmentalization of bead bound Streptavidin DNA and subsequent production and linkage of the gene product (genotype-phenotype linkage). Step 3: Assembly of the artificial metalloenzyme and encapsulation into water-in-oil-in-water droplets. Step 4: Screening for highly active enzymes able to perform new-to-nature ring closing metathesis reaction.
  • A: Encapsulation of an aqueous phase using a microfluidic chip.
B: Mono disperse droplets containing a bead bound DNA and cell-free extract for CFPS.
C: Encapsulation of a W/O emulsion into a second aqueous phase.
D: Water-in-oil-in-water droplets harboring the first CFX containing droplets.
    A: Encapsulation of an aqueous phase using a microfluidic chip. B: Mono disperse droplets containing a bead bound DNA and cell-free extract for CFPS. C: Encapsulation of a W/O emulsion into a second aqueous phase. D: Water-in-oil-in-water droplets harboring the first CFX containing droplets.
  • Compartmentalized cell free extract for the production of sfGFP.
    Compartmentalized cell free extract for the production of sfGFP.

Synthetic systems provide substantial degrees of freedom, but so far, lack complex functionality. Research methods to efficiently assemble increasingly multifunctional in vitro systems and interface them with hybrid environments to engineer novel, new-to-nature functionalities will be the main goal of this project.

Living cells are usually too complex and their internal network members too interdependent to be tolerant of major functional rearrangements. This project will use the flexibility of compartmentalized synthetic systems to functionally characterize transcriptional and translational signals and provide tools to fine-tune the composition of the cell free extract on which the assembly of the synthetic system is based.

The use of hybrid chemical/biological systems is still a seriously underexplored area. Groundbreaking examples of the flexibility of this approach have been delivered by the group lead by Wolfgang Meier. Other examples include cell free protein synthesis (CFPS), multi-step catalysis, regulation, and minimal cells. Further advances require methods for the controlled assembly of more complex systems and for exploiting the additional flexibility of molecular systems towards new-to-nature (e.g. chemical) system components.

The targeted, complex, multi-component molecular systems are based largely on biological components that need to be recruited and assembled. To prevent laborious one-by-one purification, an inverse strategy will be followed starting with a cell free extract (CFX) from a fully engineered bacterial cell.

In nature, operons are a common bacterial organization principle for functional gene clusters. There, the genes are encoded on a single transcript and protein expression levels are modulated via different control elements. In order to be able to engineer those bacterial systems in a predictive manner, design rules for synthetic multi-gene expression systems need to be established. To achieve the deduction of those rules, we intend to construct thousands of such operons varying different control elements and subsequently quantify protein products and mRNA content. Ultimately, this should allow pre-programming molecular systems at the computer.

As an expansion to on-going efforts to use insulated synthetic molecular systems for preparative chemistry, an approach to optimizing CFPS (Fig. d) will be applied. CFPS is essential in a large variety of evolution efforts that are based on or are supposed to lead to hybrid systems. The focus will be on removing interfering activities such as ATPases, termination factors, and amino acid degrading enzymes while implementing efficient energy regeneration systems.

Later, flexibility of synthetic molecular systems to expand classical protein biochemistry by including non-canonical amino acids into synthesized proteins will be exploited (Fig. e), opening up hosts of possibilities of interfacing novel proteins with synthetic environments (recruitment into membranes, immobilization on functionalized surfaces, complex building) or with novel functionalities (novel cofactors, additional catalytic flexibility).

Articles

M. Jeschek, R. ReuterT. Heinisch, C. Trindler, J. Klehr, S. PankeT. R. WardDirected evolution of artificial metalloenzymes for in vivo metathesis“ Nature doi:10.1038/nature19114 (2016). [Link] [More Information]
P. RottmannT. R. WardS. PankeCompartmentalization – A Prerequisite for Maintaining and Changing an Identity“ Chimia 6, 428 (2016).
M. Jeschek, D. GerngrossS. PankeRationally reduced libraries for combinatorial pathway optimization minimizing experimental effort“ Nat. Commun. 7, doi:10.1038/ncomms11163 (2016). [Link]

Who works with whom?

Prof. Sven Panke of the ETH Zürich at Basel (Dept. of Biosystems Science and Engineering) leads this project and works with PhD students Philipp Rottmann and Daniel Gerngross.

Group

Read more about the Panke-Group here.

Collaborations

The basis for molecular factories will be developed in collaboration with projects lead by Thomas R. Ward, Jörg Stelling, Yaakov Benenson and Wolfgang Meier.